Methylation modification-based tumor marker stamp-ep1

ABSTRACT

A methylated tumor marker STAMP-EP1 and a use thereof, which relate to the field of molecular biology. The present disclosure further relates to a use of the methylated tumor marker STAMP-EP1 in the preparation of tumor diagnostic agents. The tumor marker STAMP-EP1 herein is hypermethylated in all tumor types, is hypomethylated in corresponding normal tissue, and with very high sensitivity and specificity; primers for detecting STAMP-EP1 may be used to prepare a tumor diagnostic kit.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing in ASCIItext file (Name: 4790_0010001_Seqlisting_ST25; Size: 16,306 bytes; andDate of Creation: Apr. 22, 2021) is herein incorporated by reference inits entirety.

FIELD OF DISCLOSURE

The disclosure is in the field of disease diagnostic markers. Morespecifically, the disclosure relates to a methylation based tumor markerSTAMP, Specific Tumor Aligned Methylation of Pan-cancer.

BACKGROUND OF DISCLOSURE

The occurrence and development of tumor is a complex, multi-level,multi-factor dynamic process, including the complex interaction ofexternal environment, genetic variations and epigenetic changes, etc.Environmental factors include carcinogenic physical, chemical,biological and other factors as well as unhealthy living habits. Geneticvariations include gene mutation, copy number variation, chromosometranslocation and so on. Epigenetic changes mainly include DNAmethylation, histone modification, non-coding RNA and other factors. Inthe process of tumor occurrence and development, environmental, geneticand epigenetic factors complement each other and act together, leadingto a series of inactivations of tumor suppressor genes and activationsof proto-oncogenes, thereby causing tumor. The three factors actthroughout the development of tumor and interact with each other. Forhuman beings, there are still many challenges for tumor therapy atpresent. Although new surgical methods, targeted therapy andimmunotherapy have made some gratifying progress in recent years, thereare still many misconceptions about tumor. The major problems of tumormetastasis, recurrence, heterogeneity and drug resistance need to besolved urgently.

There are many types of tumors in human body, and the occurrences oftumors can be found in most of the tissues. Different types of tumorscan be divided into many subtypes. Especially in recent years, theclassification of tumors is more detailed due to the progress in tumormolecular biology. For different types, different stages or differentmolecular subtypes of tumors, the therapeutic regimes are alsodifferent.

With the deepening of the understanding in tumor and the progress ofscience and technology, many new tumor markers have been found and usedin clinical diagnosis. Before 1980, tumor markers were mainly hormones,enzymes, proteins and other cell secretions, such as carcinoembryonicantigen (CEA) and alpha fetoprotein (AFP) used as markers of gastriccancer, liver cancer and other tumors, carbohydrate antigen 125 (CA125)used as a marker of cervical cancer, and prostate specific antigen (PSA)used as a marker of prostate cancer. Although these tumor markers arestill used in clinic, their sensitivity and accuracy can not meet theclinical needs.

Fluid biopsy is a technique for the diagnosis and prediction of tumorsusing circulating tumor cells or circulating tumor DNA as detectiontargets. The technology is still in its infancy, having manyshortcomings. First, the sensitivity and specificity are not goodenough. The tumor itself is heterogeneous, including a variety ofsubtypes of cell populations. The proportion of tumor DNA in clinicalsamples, especially blood samples, is very low. The existing tumormarkers are difficult to meet the sensitivity of clinical requirements,and it is easy to cause misdiagnosis. Second, one marker has good effectonly for one or a few kinds of tumors. As the DNA in blood are verycomplex, the existing tumor markers cannot solve the complex problems oftumor source and metastasis. Because of these complexities, it isdifficult for many DNA methylation tumor markers to have a unifiedstandard in clinical application, which seriously affects thesensitivity and accuracy of the markers. Human tumors have bothcharacteristics and commonness. A common marker for different tumors isof great significance for tumor screening, diagnosis, treatment andefficacy evaluation.

Therefore, it is urgent to develop new tumor markers with generalapplicability, high accuracy and allowing easy judgment in tumordiagnosis.

SUMMARY OF DISCLOSURE

The object of the disclosure is to provide a method for detecting tumorbased on abnormal hypermethylation of specific sites in tumor using DNAmethylation modification as tumor marker.

The first aspect of the present disclosure provides an isolatedpolynucleotide, including: (a) a polynucleotide with a nucleotidesequence as shown in SEQ ID NO: 1; (b) a fragment of the polynucleotideof (a), having at least one (such as 2-178, more specifically 3, 5, 10,15, 20, 25, 30, 50, 60, 80, 100, 120, 150) CpG site with modification;and/or (c) a nucleic acid (such as the polynucleotide with a nucleotidesequence as shown in SEQ ID No: 3) complementary to the polynucleotideor fragment of (a) or (b).

In a preferable embodiment, said modification includes 5-methylation,5-hydroxymethylation, 5-formylcytosine (5fC) or 5-carboxylcytosine(5-caC).

The second aspect of the disclosure provides an isolated polynucleotide,which is converted from the polynucleotide of the first aspect, and ascompared with the sequence of the first aspect, the cytosine C of theCpG site(s) with modification is unchanged, and the unmodified cytosineis converted into T or U.

In a preferable embodiment, it is converted from the polynucleotidecorresponding to the first aspect by bisulfite treatment. In anotherpreferable embodiment, the polynucleotide includes: (d) a polynucleotidewith a nucleotide sequence as shown in SEQ ID NO: 2 or 4; (e) a fragmentof the polynucleotide of (d), having at least one (such as 2-178, morespecifically 3, 5, 10, 15, 20, 25, 30, 50, 60, 80, 100, 120, 150) CpGsite with modification.

The third aspect of the disclosure provides a use of the polynucleotidedescribed in the first or second aspect in manufacture of a tumordetection agent or kit.

In a preferable embodiment, the tumors include (but are not limited to):hematologic cancers such as leukemia, lymphoma, multiple myeloma;digestive system tumors such as esophageal cancer, gastric cancer,colorectal cancer, liver cancer, pancreatic cancer, bile duct andgallbladder cancer; respiratory system tumors such as lung cancer,pleuroma; nervous system tumors such as glioma, neuroblastoma,meningioma; head and neck tumors such as oral cancer, tongue cancer,laryngeal cancer, nasopharyngeal cancer; gynecological and reproductivesystem tumors such as breast cancer, ovarian cancer, cervical cancer,vulvar cancer, testicular cancer, prostate cancer, penile cancer;urinary system tumors such as kidney cancer, bladder cancer, skin andother systems tumors such as skin cancer, melanoma, osteosarcoma,liposarcoma, thyroid cancer.

In another preferable embodiment, samples of the tumor include but arenot limited to: tissue samples, paraffin embedded samples, bloodsamples, pleural effusion samples, and alveolar lavage fluid samples,ascites and lavage fluid samples, bile samples, stool samples, urinesamples, saliva samples, sputum samples, cerebrospinal fluid samples,cell smear samples, cervical scraping or brushing samples, tissue andcell biopsy samples.

The fourth aspect of the disclosure provides a method of preparing atumor detection agent, including: providing the polynucleotide describedin the first or second aspect, designing a detection agent forspecifically detecting the modification on CPG site(s) of a targetsequence which is the full length or fragment of the polynucleotide;wherein, the target sequence has at least one (such as 2-178, morespecifically, 3, 5, 10, 15, 20, 25, 30, 50, 60, 80, 100, 120, 150)modified CpG site; preferably, the detection agent includes (but is notlimited to) primers or probes.

The fifth aspect of the disclosure provides an agent or a combination ofagents which specifically detect the modification on CPG site(s) of atarget sequence, which is the full length or fragment of any of thepolynucleotides described in the first or second aspect and has at leastone (such as 2-178, more specifically, 3, 5, 10, 15, 20, 25, 30, 50, 60,80, 100, 120, 150) modified CpG site.

In a preferable embodiment, the agent or combination of agents is for agene sequence containing the target sequence (designed based on the genesequence), and the gene sequence includes gene Panels or gene groups.

In another preferable embodiment, the detection agent comprises: primersor probes.

In another preferable embodiment, the primers are: the primers shown inSEQ ID NO: 3 and 4; the primers shown in SEQ ID NO: 7 and 8; the primersshown in SEQ ID NO: 9 and 10; the primers shown in SEQ ID NO: 11 and 12;or the primers shown in SEQ ID NO: 13 and 14.

In the sixth aspect of the disclosure, a use of the agent or combinationof agents described in the fifth aspect of the disclosure in themanufacture of a kit for detecting tumors is provided; preferably, thetumors include (but are not limited to): digestive system tumors such asesophageal cancer, gastric cancer, colorectal cancer, liver cancer,pancreatic cancer, bile duct and gallbladder cancer; respiratory systemtumors such as lung cancer, pleuroma; hematologic cancers such asleukemia, lymphoma, multiple myeloma; gynecological and reproductivesystem tumors such as breast cancer, ovarian cancer, cervical cancer,vulvar cancer, testicular cancer, prostate cancer, penile cancer;nervous system tumors such as glioma, neuroblastoma, meningioma; headand neck tumors such as oral cancer, tongue cancer, laryngeal cancer,nasopharyngeal cancer; urinary system tumors such as kidney cancer,bladder cancer, skin and other systems tumors such as skin cancer,melanoma, osteosarcoma, liposarcoma, thyroid cancer.

The seventh aspect of the present disclosure provides a detection kit,comprising container(s) and the agent or combination of agents describedabove in the container(s); preferably, each agent is placed in anindependent container.

In a preferable embodiment, the kit also includes: bisulfite, DNApurification agent, DNA extraction agent, PCR amplification agent and/orinstruction for use (indicating operation steps of the detection and aresult judgment standard).

In the eighth aspect of the disclosure, a method for detecting themethylation profile of a sample in vitro is provided, including: (i)providing the sample and extracting the nucleic acid; (ii) detecting themodification on CPG site(s) of a target sequence in the nucleic acid of(i), wherein the target sequence is the polynucleotide described in thefirst aspect or the polynucleotide converted therefrom, which describedin the second aspect.

In a preferable embodiment, in step (3), the analysis methods includepyrosequencing, bisulfite conversion sequencing, method usingmethylation chip, qPCR, digital PCR, second generation sequencing, thirdgeneration sequencing, whole genome methylation sequencing, DNAenrichment detection, simplified bisulfite sequencing technology, HPLC,MassArray, methylation specific PCR (MSP), or their combination, as wellas in vitro detection and in vivo tracer detection for the combined genegroup of partial or all of the methylation sites in the sequence shownin SEQ ID NO: 1. In addition, other methylation detection methods andnewly developed methylation detection methods in the future can beapplied to the disclosure.

In another preferable embodiment, step (ii) includes: (1) treating theproduct of (i) to convert the unmodified cytosine into uracil;preferably, the modification includes 5-methylation,5-hydroxymethylation, 5-formylcytosine (5fC) or 5-carboxylcytosine(5-caC); preferably, treating the nucleic acid of step (i) withbisulfite; and (2) analyzing the modification of the target sequence inthe nucleic acid treated by (1).

In another preferable embodiment, the abnormal methylation profile isthe high level of methylation of C in CPG(s) of the polynucleotide.

In another preferable embodiment, the methylation profile detectingmethod is not for the purpose of directly obtaining the diagnosis resultof a disease, or is not a diagnostic method.

The ninth aspect of the disclosure provides a tumor diagnosis kit,including primer pairs designed based on the sequence described in thefirst or second aspect of the disclosure, and gene Panels or gene groupscontaining the sequence, to obtain the characteristics of normal cellsand tumor cells through DNA methylation detection.

Other aspects of the disclosure will be apparent to those skilled in theart based on the disclosure herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the average methylation value of STAMP-EP1 in the genomicDNA of lung cancer cell line A549, with the corresponding value of thegenomic DNA of normal lung fibroblast cell line MRC5 as the control.Dark squares indicate the corresponding sites “methylated”, while lightsquares indicate the corresponding sites “non-methylated”.

FIG. 2 shows comparison of STAMP-EP1 methylation value (left), thedetection specificity and sensitivity (right) between control group andexperimental group of 20 pairs of paracancerous-lung cancer samples,with paracancerous samples as lung cancer control group, and lung cancersamples as lung cancer experimental group.

FIG. 3. 10 paracancerous clinical samples of colorectal cancer were usedas the control group, and 28 colorectal cancer clinical samples wereused as the experimental group. The STAMP-EP1 methylation value (left),the detection specificity and sensitivity (right) were compared betweenthe control group and the experimental group.

FIG. 4. 10 normal gastric (or gastritis) clinical samples were used asthe control group, 10 gastric cancer resection edge clinical sampleswere used as the experimental group 1, and 20 gastric cancer clinicalsamples were used as experimental group 2. The STAMP-EP1 methylationvalue was compared among the three groups.

FIG. 5. 13 paracancerous clinical samples of cervical cancer were usedas the control group, and 26 cervical cancer clinical samples were usedas the experimental group. The STAMP-EP1 methylation value (left), thedetection specificity and sensitivity (right) were compared between thecontrol group and the experimental group.

FIG. 6 shows comparison of STAMP-EP1 methylation value (left), thedetection specificity and sensitivity (right) between control group andexperimental group of 21 pairs of paracancerous-liver cancer samples,with paracancerous samples as control group, and liver cancer samples asexperimental group.

FIG. 7 shows comparison of STAMP-EP1 methylation value (left), thedetection specificity and sensitivity (right) between control group andexperimental group of 22 pairs of paracancerous-breast cancer samples,with paracancerous samples as control group, and breast cancer samplesas experimental group.

FIG. 8 shows comparison of STAMP-EP1 methylation value (left), thedetection specificity and sensitivity (right) between control group andexperimental group of 18 pairs of paracancerous-pancreatic cancersamples, with paracancerous samples as control group, and pancreaticcancer samples as experimental group.

FIG. 9. Eleven pairs of paracancerous-head and neck cancer samples wereobtained, including 5 cases of laryngeal cancer, 2 cases of tonsilcancer, 2 cases of epiglottis cancer, 1 case of tongue base cancer, and1 case of hypopharyngeal cancer. STAMP-EP1 methylation value wascompared between head and neck cancer control group and experimentalgroup, with paracancerous samples as control group, and cancer samplesas experimental group.

FIG. 10 shows comparison of STAMP-EP1 methylation value (left), thedetection specificity and sensitivity (right) between control group andexperimental group of bile samples from 12 non-cancer patients and 10gallbladder cancer patients, with non-cancer samples as control group,and gallbladder cancer samples as experimental group.

FIG. 11. Ten non-leukemia bone marrow smear samples were used as thecontrol group, and 20 leukemia bone marrow smear samples were used asthe experimental group. The STAMP-EP1 methylation value (left), thedetection specificity and sensitivity (right) were compared between thecontrol group and experimental group.

FIG. 12. Eight paracancerous clinical samples of renal cancer were usedas the control group, and 16 renal cancer clinical samples were used asthe experimental group. The STAMP-EP1 methylation value (left), thedetection specificity and sensitivity (right) were compared between thecontrol group and experimental group.

FIG. 13. Five cases of bladder cancer paracancerous samples andnon-cancer urine samples were used as the control group, and 7 cases ofbladder cancer tissue samples and bladder cancer urine samples were usedas the experimental group. The STAMP-EP1 methylation value was comparedbetween the control group and the experimental group.

FIG. 14. Twenty normal human plasma samples were used as the controlgroup, and plasma samples from patients with different tumor types wereobtained, including 10 cases of liver cancer, 10 cases of pancreaticcancer, 10 cases of lung cancer, 10 cases of colorectal cancer and 10cases of breast cancer. The STAMP-EP1 methylation value was comparedbetween the control group and the experimental group.

DETAILED DESCRIPTION

The inventor is committed to the research of tumor markers. Afterextensive research and screening, the inventor provides a universal DNAmethylation tumor marker, STAMP (Specific Tumor Aligned Methylation ofPan-cancer). In normal tissues, STAMP was hypomethylated, while in tumortissues, it was hypermethylated. It can be used for clinical tumordetection and as the basis of designing tumor diagnostic agents.

Term

As used herein, “isolated” refers to a material separated from itsoriginal environment (if the material is a natural material, theoriginal environment is the natural environment). For example, in livingcells, polynucleotides and polypeptides in their natural state are notisolated or purified, but the same polynucleotides or polypeptides willbe isolated ones if they are separated from other substances existed inthe natural state.

As used herein, “sample” includes substances suitable for DNAmethylation detection obtained from any individual or isolated tissue,cell or body fluid (such as plasma). For example, the samples includebut are not limited to: tissue samples, paraffin embedded samples, bloodsamples, pleural effusion samples, and alveolar lavage fluid samples,ascites and lavage fluid samples, bile samples, stool samples, urinesamples, saliva samples, cerebrospinal fluid samples, cell smearsamples, cervical scraping or brushing samples, tissue and cell biopsysamples.

As used herein, “hypermethylation” refers to high level of methylation,hydroxymethylation, 5-formylcytosine (5fC) or 5-carboxylcytosine (5-caC)of CpG in a gene sequence. For example, in the case of methylationspecific PCR (MSP), if the PCR reaction with methylation specificprimers has positive PCR results, the DNA (gene) region of interest isin hypermethylation state. For another example, in the case of real-timequantitative methylation specific PCR, hypermethylation can bedetermined based on statistic difference of the methylation status valueas compared with the control sample.

As used herein, the tumors include but are not limited to: hematologiccancers such as leukemia, lymphoma, multiple myeloma; digestive systemtumors such as esophageal cancer, gastric cancer, colorectal cancer,liver cancer, pancreatic cancer, bile duct and gallbladder cancer;respiratory system tumors such as lung cancer, pleuroma; nervous systemtumors such as glioma, neuroblastoma, meningioma; head and neck tumorssuch as oral cancer, tongue cancer, laryngeal cancer, nasopharyngealcancer; gynecological and reproductive system tumors such as breastcancer, ovarian cancer, cervical cancer, vulvar cancer, testicularcancer, prostate cancer, penile cancer; urinary system tumors such askidney cancer, bladder cancer, skin and other systems tumors such asskin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer.

Gene Marker

In order to find a useful target for tumor diagnosis, the inventor hasidentified the target of STAMP-EP1 after extensive and in-depthresearch. The methylation status of the sequence of STAMP-EP1 gene issignificantly different between tumor tissues and non-tumor tissues. Ifthe abnormal hypermethylation of the sequence of STAMP-EP1 gene of asubject is detected, the subject can be identified as having a high-riskof tumor. Moreover, the significant difference of STAMP-EP1 betweentumor and non-tumor tissues exists in different types of tumors,including solid tumors and non-solid tumors.

Therefore, the disclosure provides an isolated polynucleotide,comprising the nucleotide sequence shown in the sequence of SEQ ID NO: 1or SEQ ID NO: 3 (the reverse complementary sequence of SEQ ID NO: 1).For tumor cells, the polynucleotide contains 5-methylcytosine (5mC) at Cpositions of many 5′-CpG-3′. The disclosure also comprises fragments ofthe polynucleotide of the sequence shown in SEQ ID NO: 1 or 3, having atleast one (such as 2-178, more specifically 3, 5, 10, 15, 20, 25, 30,50, 60, 80, 100, 120, 150) methylated CpG site. The abovepolynucleotides or fragments can also be used in the design of detectionagents or detection kits.

In some specific embodiments of the disclosure, the fragments of thepolynucleotide are, for example, a fragment containing the residues1-589 of SEQ ID NO: 1 (containing CpG sites 001-059); a fragmentcontaining the residues 632-1218 of SEQ ID NO: 1 (containing CpG sites060-100); a fragment containing the residues 1322-2066 of SEQ ID NO: 1(containing CpG sites 101-147); a fragment containing the residues2100-2448 of SEQ ID NO: 1 (containing CpG sites 148-178). Antisensechains of the above fragments are suitable for use in the disclosure.These fragments are merely examples of preferable embodiments of thepresent disclosure. Based on the information provided by the presentdisclosure, other fragments can also be selected.

In addition, gene Panels or gene groups containing the sequence shown inthe SEQ ID NO: 1 or SEQ ID NO: 2 or fragments thereof are alsoencompassed by the disclosure. For the gene Panel or gene group, thecharacteristics of normal cells and tumor cells can also be identifiedthrough DNA methylation detection.

The above polynucleotides can be used as the key regions for analysis ofthe methylation status in the genome. Their methylation status can beanalyzed by various technologies known in the art. Any technique thatcan be used to analyze the methylation state can be applied to thepresent disclosure.

When treated with bisulfite, un-methylated cytosine(s) of the abovepolynucleotides will be converted into uracil, while methylatedcytosine(s) remained unchanged.

Therefore, the disclosure also provides the polynucleotides obtainedfrom the above polynucleotides (including the complementary chain(antisense chain)) after being treated with bisulfite, including thepolynucleotides of the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.These polynucleotides can also be used in the design of detection agentsor detection kits.

The disclosure also comprises fragments of the polynucleotides obtainedfrom the above polynucleotides or the antisense chain thereof afterbeing treated with bisulfite, wherein the fragments contain at least onemethylated CpG site.

Detection Agents and Kits

Based on the new discovery of the disclosure, a detection agent designedbased on said polynucleotide(s) is also provided for detecting themethylation profile of polynucleotide(s) in the sample in vitro. Thedetection methods and agents known in the art for determining thesequence, variation and methylation of the genome can be applied in thedisclosure.

Therefore, the disclosure provides a method of preparing a tumordetection agent, including: providing the polynucleotide, designing adetection agent for specifically detecting a target sequence which isthe full length or fragment of the polynucleotide; wherein, the targetsequence has at least one methylated CpG site.

The detection agent herein includes but is not limited to: primers,probes, etc.

For example, the agent is primer pairs. Based on the sequence of thepolynucleotide, those skilled in the art know how to design primer(s).The two primers are on each side of the specific sequence of the targetgene to be amplified (including CpG sequence, for the gene regionoriginally methylated, the primer is complementary with CpG, and for thegene region originally un-methylated, the primer is complementary withTpG). It should be understood that based on the new discovery of thedisclosure, those skilled in the art can design a variety of primers orprobes or other types of detection agents for CpG sites at differentpositions on the target sequence or their combinations. These primers orprobes or other types of detection agents should be included in thetechnical solution of the present disclosure.

The agent can also be a combination of agents (primer combination),including more than one set of primers, so that the multiplepolynucleotides can be amplified respectively.

The disclosure also provides a kit for detecting the methylation profileof polynucleotide in a sample in vitro, which comprises container(s) andthe above primer pair(s) in the container(s).

In addition, the kit can also include various reagents required for DNAextraction, DNA purification, PCR amplification, etc.

In addition, the kit can also include an instruction for use, whichindicates operation steps of the detection and a result judgmentstandard, for the application of those skilled in the art.

Detection Method

The methylation profile of a polynucleotide can be determined by anytechnique in the art (such as methylation specific PCR (MSP) orreal-time quantitative methylation specific PCR, Methylight), or othertechniques that are still developing and will be developed.

Quantitative methylation specific PCR (QMSP) can also be used to detectmethylation level. It is a continuous optical monitoring method based onfluorescent PCR, which is more sensitive than MSP. It has highthroughput and avoids electrophoresis based result analysis.

Other available technologies include conventional methods in the artsuch as pyrosequencing, bisulfite converstion sequencing, qPCR, secondgeneration sequencing, whole genome methylation sequencing, DNAenrichment detection, simplified bisulfite sequencing or HPLC, andcombined gene group detection. It should be understood that, on thebasis of the new disclosure herein, these well-known technologies andsome technologies to be developed in the art can be applied to thepresent disclosure.

As a preferable embodiment of the disclosure, a method of detecting themethylation profile of a polynucleotide in a sample in vitro is alsoprovided. The method is based on the follow principle: the un-methylatedcytosine can be converted into uracil by bisulfite, which can betransformed into thymine in the subsequent PCR amplification process,while the methylated cytosine remains unchanged; therefore, after thepolynucleotide is treated by bisulfite, the methylated site presents apolynucleotide polymorphism (SNP) similar to C/T. Based on the aboveprinciple, methylated and un-methylated cytosine can be distinguishedeffectively by identifying the methylation profile of a polynucleotidein the sample.

The method of the disclosure includes: (a) providing samples andextracting genomic DNA; (b) treating the genomic DNA of step (a) withbisulfite, so as to convert the un-methylated cytosine in the genomicDNA into uracil; (c) analyzing whether the genomic DNA treated in step(b) contains an abnormal methylation profile.

The method of the disclosure can be used for: (i) analyzing whether asubject has tumor by detecting the sample of the subject; (ii)identifying a population having high-risk of tumor. The method needs notto be aimed at obtaining direct diagnosis results.

In a preferable embodiment of the disclosure, DNA methylation isdetected by PCR amplification and pyrosequencing. It should beunderstood by those in the art that DNA methylation detection is notlimited to these methods, and any other DNA methylation detection methodcan also be used. The primers used in PCR amplification are not limitedto those provided in Examples.

Because of bisulfite treatment, in which un-methylated cytosine ingenomic DNA are converted into uracil and then transformed into thyminein the subsequent PCR process, the sequence complexity of the genomewill be reduced, and it will be more difficult to amplify specifictarget fragments by PCR. Therefore, in order to improve amplificationefficiency and specificity, nested PCR may be preferable, wherein twosets of primers (outer primers and inner primers) are used in twosuccessive runs of PCR, and the amplification product from the first runundergoes a second run with the second set of primers. However, itshould be understood that the detection methods suitable for the presentdisclosure are not limited thereto.

After the research and verification on clinical samples, the method ofthe disclosure provides very high accuracy in the clinical diagnosis oftumors. The disclosure can be applied to the fields of tumor auxiliarydiagnosis, efficacy evaluation, prognosis monitoring, etc., thus has ahigh clinical value.

The disclosure is further illustrated by the specific examples describedbelow. It should be understood that these examples are merelyillustrative, and do not limit the scope of the present disclosure. Theexperimental methods without specifying the specific conditions in thefollowing examples generally used the conventional conditions, such asthose described in J. Sambrook, Molecular Cloning: A Laboratory Manual(3rd ed. Science Press, 2002) or followed the manufacturer'srecommendation.

Example 1. Nucleic Acid Sequence for STAMP-EP1 Detection

The sequence of the STAMP-EP1 tumor marker is provided as follows: SEQID NO: 1 (chr14: 60975733 bp˜60978180 bp (hg19/Human)), in which theunderlined bases are methylated CpG sites, and each number below theunderline indicates the site number.

   1~50 bp CGGCGGGCGC TGTCGAGCAC GGGGAGGTGC TGAAATAGTC CTGGCGTGCT001 002 003   004 005                           006   51~100 bpGATTCAAGCT TTGATTGGCA GAGCCACCCG GTGACTGACA GGGGGTCTCC                             007  101~150 bpATGGCGCCCG CGCCGCCAAT CCGCCCACCC CAATAGCGGA GCCAGCTCGC   008 009 010 011     012             013        014  151~200 bpCTGCCGGCGT GCCTGAGCCG AGCCGAGCCC GAACCCCAAG CCGCGGAGCC   015 016        017    018 019            020 021  201~250 bpAGCACCTCCT CCAGTCGGGG TCGTCCGCTC CCGGCCGTTG AGCCACCGCC                022   023  024   025  026        027 028  251~300 bpGCCACCCGGT AGTGTGTCCC GCTGCCCCAA TCCGCCTCAT CAACAAGCGC      029         030              031             032  301~350 bpCTGGCACACT CAGCCAGGCC CGCGGGCATC TGCTGCGTGT CCCGCTCCGG                      033 034         035    036   037  351~400 bpGCTCAGTGCC CTCGCCGCCG CCGGCACTGC CTCGATGTTC CAGCTGCCCA           038 039 040 041         042  401~450 bpTCTTGAATTT CAGCCCCCAG CAAGTGGCCG GGGTATGTGA GACCCTGGAA                             043  451~500 bpGAGAGCGGCG ATGTGGAGCG CCTGGGTCGC TTCCTCTGGT CGCTGCCCGT    044 045       046       047             048   049  501~550 bpGGCCCCTGCG GCCTGCGAGG CCCTCAACAA GAATGAGTCG GTGCTACGCG       050     051                      052      053 054  551~600 bpCACGAGCCAT CGTGGCCTTT CACGGTGGCA ACTACCGCGA GCTCTATCAT 055       056         057          058 059  601~650 bpATCCTGGAAA ACCACAAGTT CACCAAGGAG TCGCACGCCA AGCTGCAGGC                                 060  061            062  651~700 bpGCTGTGGCTT GAAGCACACT ACCAGGAGGC TGAGAAGCTG CGTGGAAGAC                                            063  701~750 bp

 751~800 bp

 801~850 bp GCGGCACCTG CTACGCGAGT GGTACCTGCA GGATCCATAC CCTAACCCCA 072         073 074  851~900 bpGCAAAAAACG TGAGCTCGCC CAGGCAACCG GACTGACCCC TACGCAGGTG       075      076          077              078  901~950 bpGGCAACTGGT TCAAAAACCG CCGACAAAGG GACCGAGCGG CTGCAGCCAA                 079   080          081 082  951~1000 bpGAACAGGTCG GTACCTAGAG GCCTCCGCGC TTTGAGCGCA CCGGGGAGGA       083               084 085       086  087 1001~1050 bpGGCGGGTGGA GGCACCTCTG GCGCCCTTAC CCAGTCCCTG GCGACTCCAA  088                  089                   090 1051~1100 bpTTCAGCAGGA GTTGGGAGCG CGGTCTGTCT TGGGTTAAGA GCCCTGCGTT                  091 092                         093 1101~1150 bpCTGGGCTCCT GGCCGGGAGT TCCCTTGCCG GCTCTGCTTC CCCACCCGCT              094             095                 096 1151~1200 bpGGCTCCCCAC GCCTGCGGGC AGCTGCAGCA GCTGGTCCCG GTCACCAAAC       097     098                      099 1201~1250 bpCAAGGCTTCA CTGGGACGGA GAGGGGAAGA GAAATAAAAA ATTAAAATCC                 100 1251~1300 bpTACAAACAGT TAGGGACCCC AAGACCCAAA GCTAATTCTT GTCAGCCTGG 1301~1350 bpGCACAGGCTC CTACTATTAA TCGAAGCCTG GCTTATTAGC AATGTGTCGG                      101                         102 1351~1400 bpTTTCATGTTA ATTATCATTT TCAAAGCCCA GGTATATCCC TCCCTAATGC 1401~1450 bpTTTGAAAACA GTTTTCAATG GACTTTTGAG AAATGGGAAG TCGAGTTTTC                                             103 1451~1500 bpCTCTTCCCAT GCGCTGCCTG CCACTCTTGT CTCAAAACAG CAAACTAGTC             1041501~1550 bp CGTGGGCCGA GGCTTTTCGT TTCCCGGAGT GTGGATCTCG ATTAGCCAAA105    106        107     108            109 1551~1600 bpCATTTTGCGG AAGAGCCCGG CCTCATCCCC CAGGCCCAAA TGCTCCTTAC       110        111 1601~1650 bpAATCCTTTTT GCCTTTAGGT CGGGCCGACC CGATCCAACG CGATCGCGGG                      112  113   114    115 116 117 118 1651~1700 bpAGCACTTGCT CAGGCGTAAG CCCCAGGCAG ACGCACCGTT AGAAATGGTA              119                 120  121 1701~1750 bpTCCCATGTCC CTGGGACCGA TCTGTCCTTG TCACCCACAC TTCGTTTATT                  122                         123 1751~1800 bpTCCTGACAGT CCTGTAAATC TCCCAAAAGT GCACAACAAA CAGGGAGGAC 1801~1850 bpACTGCAAGCC CAGTATATAA AAGACCTGGG AGCTGCGGCG CTGAGAAAGG                                     124 125 1851~1900 bpGCGCGAATCA TGGTGGGGCA CAACAGTAGG GACCCGCGGA GGGGCGGCCG126 127                             128 129    130 131 1901~1950 bpCGGACTCCTG CCCGACCTCT GTCGCCTTGC CGAGTAATCC TCGCCTTAAC132         133        134       135        136 1951~2000 bpTGCTGGGGTC TTCGGAAGAA CCTCTAGCCG CCGGGCTGGA GGGACGCAGG            137              138  139           140 2001~2050 bpAGGTGGTGGG GGCGGGCGAC GGGCGGCTGT GTTACGAGCT GTGACCCGTG           141 142 143   144         145          146 2051~2100 bpTTCCCTTTCT TCCCCGTAGA CTCCAGCAGC AGGTCCTGTC ACAGGGTTCC              147                                    148 2101~2150 bpGGGCGGGCAC TACGGGCGGA GGGCGACGGC ACGCCAGAGG TGCTGGGCGT  149       150  151     152 153  154              155 2151~2200 bpCGCCACCAGC CCGGCCGCCA GTCTATCCAG CAAGGCGGCC ACTTCAGCCA156         157 158                   159 2201~2250 bpTCTCCATCAC GTCCAGCGAC AGCGAGTGCG ACATCTGAGT TGCCCATCCA        160      161    162   163 2251~2300 bpGGATGCTCAG AAGCAGATTC CAGTGTAAAA ACGAGAAAAA CAAAATGAAA                                  164 2301~2350 bpGAGGGGAAGA AGATGAGAGA CCTGCAAATC CAGCGCCACA GAAGCCAGGT                                    165 2351~2400 bpGACCAGGGAC CCGCGGGCTC GGGTTGCCGT TTCCCGCCCC ACCCCGCGGC           166 167 168       169     170      171 172 2401~2448 bpCGGCCTGGCT TCACTGGCGC CCTTTGGCCG CGACCACGGG AACCAGCG173               174        175 176   177       178

The bisulfite treated sequence from SEQ ID NO: 1 is shown in SEQ ID NO:2 (Y represents C or U) as follows:

    1~50 bp YGGYGGGYGU TGTYGAGUAY GGGGAGGTGU TGAAATAGTU UTGGYGTGUT001 002003    004 005                           006   51~100 bpGATTUAAGUT TTGATTGGUA GAGUUAUUYG GTGAUTGAUA GGGGGTUTUU                             007  101~150 bpATGGYGUUYG YGUYGUUAAT UYGUUUAUUU UAATAGYGGA GUUAGUTYGU   008 009 010 011     012             013         014  151~200 bpUTGUYGGYGT GUUTGAGUYG AGUYGAGUUY GAAUUUUAAG UYGYGGAGUU   015 016        017    018 019             020 021  201~250 bpAGUAUUTUUT UUAGTYGGGG TYGTUYGUTU UYGGUYGTTG AGUUAUYGUY                022    023 024   025  026      027 028  251~300 bpGUUAUUYGGT AGTGTGTUUY GUTGUUUUAA TUYGUUTUAT UAAUAAGYGU      029         030              031             032  301~350 bpUTGGUAUAUT UAGUUAGGUU YGYGGGUATU TGUTGYGTGT UUYGUTUYGG                      033 034         035     036 037  351~400 bpGUTUAGTGUU UTYGUYGUYG UYGGUAUTGU UTYGATGTTU UAGUTGUUUA           038 039040  041         042  401~450 bpTUTTGAATTT UAGUUUUUAG UAAGTGGUYG GGGTATGTGA GAUUUTGGAA                             043  451~500 bpGAGAGYGGYG ATGTGGAGYG UUTGGGTYGU TTUUTUTGGT YGUTGUUYGT    044045        046        047            048    049  501~550 bpGGUUUUTGYG GUUTGYGAGG UUUTUAAUAA GAATGAGTYG GTGUTAYGYG       050      051                     052    053 054  551~600 bpUAYGAGUUAT YGTGGUUTTT UAYGGTGGUA AUTAUYGYGA GUTUTATUAT  055      056          057         058 059  601~650 bpATUUTGGAAA AUUAUAAGTT UAUUAAGGAG TYGUAYGUUA AGUTGUAGGY                                  060 061          062  651~700 bpGUTGTGGUTT GAAGUAUAUT AUUAGGAGGU TGAGAAGUTG YGTGGAAGAU                                            063  701~750 bpUUUTGGGAUU TGTGGAUAAG TAUYGAGTAA GGAAGAAGTT UUYGUTGUYG                         064                  065  066  751~800 bpYGUAUUATTT GGGAYGGYGA AUAGAAGAUA UAUTGUTTUA AGGAGYGUAY067           068 069                          070 071  801~850 bpGYGGUAUUTG UTAYGYGAGT GGTAUUTGUA GGATUUATAU UUTAAUUUUA 072          073 074  851~900 bpGUAAAAAAYG TGAGUTYGUU UAGGUAAUYG GAUTGAUUUU TAYGUAGGTG       075      076          077              078  901~950 bpGGUAAUTGGT TUAAAAAUYG UYGAUAAAGG GAUYGAGYGG UTGUAGUUAA                  079  080          081 082  951~1000 bpGAAUAGGTYG GTAUUTAGAG GUUTUYGYGU TTTGAGYGUA UYGGGGAGGA       083                084 085      086   087 1001~1050 bpGGYGGGTGGA GGUAUUTUTG GYGUUUTTAU UUAGTUUUTG GYGAUTUUAA  088                  089                   090 1051~1100 bpTTUAGUAGGA GTTGGGAGYG YGGTUTGTUT TGGGTTAAGA GUUUTGYGTT                  091 092                         093 1101~1150 bpUTGGGUTUUT GGUYGGGAGT TUUUTTGUYG GUTUTGUTTU UUUAUUYGUT              094            095                  096 1151~1200 bpGGUTUUUUAY GUUTGYGGGU AGUTGUAGUA GUTGGTUUYG GTUAUUAAAU       097      098                     099 1201~1250 bpUAAGGUTTUA UTGGGAYGGA GAGGGGAAGA GAAATAAAAA ATTAAAATUU                 100  1251~1300 bpTAUAAAUAGT TAGGGAUUUU AAGAUUUAAA GUTAATTUTT GTUAGUUTGG 1301~1350 bpGUAUAGGUTU UTAUTATTAA TYGAAGUUTG GUTTATTAGU AATGTGTYGG                       101                        102 1351~1400 bpTTTUATGTTA ATTATUATTT TUAAAGUUUA GGTATATUUU TUUUTAATGU 1401~1450 bpTTTGAAAAUA GTTTTUAATG GAUTTTTGAG AAATGGGAAG TYGAGTTTTU                                             103  1451~1500 bpUTUTTUUUAT GYGUTGUUTG UUAUTUTTGT UTUAAAAUAG UAAAUTAGTU             104 1501~1550 bp YGTGGGUYGA GGUTTTTYGT TTUUYGGAGT GTGGATUTYG ATTAGUUAAA105    106        107     108           109 1551~1600 bpUATTTTGYGG AAGAGUUYGG UUTUATUUUU UAGGUUUAAA TGUTUUTTAU      110        111 1601~1650 bpAATUUTTTTT GUUTTTAGGT YGGGUYGAUU YGATUUAAYG YGATYGYGGG                      112  113   114    115 116 117118 1651~1700 bpAGUAUTTGUT UAGGYGTAAG UUUUAGGUAG AYGUAUYGTT AGAAATGGTA               119                120  121 1701~1750 bpTUUUATGTUU UTGGGAUYGA TUTGTUUTTG TUAUUUAUAU TTYGTTTATT                  122                         123 1751~1800 bpTUUTGAUAGT UUTGTAAATU TUUUAAAAGT GUAUAAUAAA UAGGGAGGAU 1801~1850 bpAUTGUAAGUU UAGTATATAA AAGAUUTGGG AGUTGYGGYG UTGAGAAAGG                                     124125 1851~1900 bpGYGYGAATUA TGGTGGGGUA UAAUAGTAGG GAUUYGYGGA GGGGYGGUYG 126127                              128129     130131 1901~1950 bpYGGAUTUUTG UUYGAUUTUT GTYGUUTTGU YGAGTAATUU TYGUUTTAAU132          133        134                  135 136 1951~2000 bpTGUTGGGGTU TTYGGAAGAA UUTUTAGUYG UYGGGUTGGA GGGAYGUAGG             137             138  139           140 2001~2050 bpAGGTGGTGGG GGYGGGYGAY GGGYGGUTGT GTTAYGAGUT GTGAUUYGTG           141 142143    144         145          146 2051~2100 bpTTUUUTTTUT TUUUYGTAGA UTUUAGUAGU AGGTUUTGTU AUAGGGTTUY               147                                 148 2101~2150 bpGGGYGGGUAU TAYGGGYGGA GGGYGAYGGU AYGUUAGAGG TGUTGGGYGT   149       150 151     152 153  154              155 2151~2200 bpYGUUAUUAGU UYGGUYGUUA GTUTATUUAG UAAGGYGGUU AUTTUAGUUA156         157 158                   159 2201~2250 bpTUTUUATUAY GTUUAGYGAU AGYGAGTGYG AUATUTGAGT TGUUUATUUA       160       161    162  163 2251~2300 bpGGATGUTUAG AAGUAGATTU UAGTGTAAAA AYGAGAAAAA UAAAATGAAA                                  164 2301~2350 bpGAGGGGAAGA AGATGAGAGA UUTGUAAATU UAGYGUUAUA GAAGUUAGGT                                    165 2351~2400 bpGAUUAGGGAU UYGYGGGUTY GGGTTGUYGT TTUUYGUUUU AUUUYGYGGU           166167 168        169     170       171 172 2401~2448 bpYGGUUTGGUT TUAUTGGYGU UUTTTGGUYG YGAUUAYGGG AAUUAGYG173               174        175 176   177       178

The reverse complementary sequence of SEQ ID NO: 1 is shown in SEQ IDNO: 3 as follows:

CGCTGGTTCCCGTGGTCGCGGCCAAAGGGCGCCAGTGAAGCCAGGCCGGCCGCGGGGTGGGGCGGGAAACGGCAACCCGAGCCCGCGGGTCCCTGGTCACCTGGCTTCTGTGGCGCTGGATTTGCAGGTCTCTCATCTTCTTCCCCTCTTTCATTTTGTTTTTCTCGTTTTTACACTGGAATCTGCTTCTGAGCATCCTGGATGGGCAACTCAGATGTCGCACTCGCTGTCGCTGGACGTGATGGAGATGGCTGAAGTGGCCGCCTTGCTGGATAGACTGGCGGCCGGGCTGGTGGCGACGCCCAGCACCTCTGGCGTGCCGTCGCCCTCCGCCCGTAGTGCCCGCCCGGAACCCTGTGACAGGACCTGCTGCTGGAGTCTACGGGGAAGAAAGGGAACACGGGTCACAGCTCGTAACACAGCCGCCCGTCGCCCGCCCCCACCACCTCCTGCGTCCCTCCAGCCCGGCGGCTAGAGGTTCTTCCGAAGACCCCAGCAGTTAAGGCGAGGATTACTCGGCAAGGCGACAGAGGTCGGGCAGGAGTCCGCGGCCGCCCCTCCGCGGGTCCCTACTGTTGTGCCCCACCATGATTCGCGCCCTTTCTCAGCGCCGCAGCTCCCAGGTCTTTTATATACTGGGCTTGCAGTGTCCTCCCTGTTTGTTGTGCACTTTTGGGAGATTTACAGGACTGTCAGGAAATAAACGAAGTGTGGGTGACAAGGACAGATCGGTCCCAGGGACATGGGATACCATTTCTAACGGTGCGTCTGCCTGGGGCTTACGCCTGAGCAAGTGCTCCCGCGATCGCGTTGGATCGGGTCGGCCCGACCTAAAGGCAAAAAGGATTGTAAGGAGCATTTGGGCCTGGGGGATGAGGCCGGGCTCTTCCGCAAAATGTTTGGCTAATCGAGATCCACACTCCGGGAAACGAAAAGCCTCGGCCCACGGACTAGTTTGCTGTTTTGAGACAAGAGTGGCAGGCAGCGCATGGGAAGAGGAAAACTCGACTTCCCATTTCTCAAAAGTCCATTGAAAACTGTTTTCAAAGCATTAGGGAGGGATATACCTGGGCTTTGAAAATGATAATTAACATGAAACCGACACATTGCTAATAAGCCAGGCTTCGATTAATAGTAGGAGCCTGTGCCCAGGCTGACAAGAATTAGCTTTGGGTCTTGGGGTCCCTAACTGTTTGTAGGATTTTAATTTTTTATTTCTCTTCCCCTCTCCGTCCCAGTGAAGCCTTGGTTTGGTGACCGGGACCAGCTGCTGCAGCTGCCCGCAGGCGTGGGGAGCCAGCGGGTGGGGAAGCAGAGCCGGCAAGGGAACTCCCGGCCAGGAGCCCAGAACGCAGGGCTCTTAACCCAAGACAGACCGCGCTCCCAACTCCTGCTGAATTGGAGTCGCCAGGGACTGGGTAAGGGCGCCAGAGGTGCCTCCACCCGCCTCCTCCCCGGTGCGCTCAAAGCGCGGAGGCCTCTAGGTACCGACCTGTTCTTGGCTGCAGCCGCTCGGTCCCTTTGTCGGCGGTTTTTGAACCAGTTGCCCACCTGCGTAGGGGTCAGTCCGGTTGCCTGGGCGAGCTCACGTTTTTTGCTGGGGTTAGGGTATGGATCCTGCAGGTACCACTCGCGTAGCAGGTGCCGCGTGCGCTCCTTGAAGCAGTGTGTCTTCTGTTCGCCGTCCCAAATGGTGCGCGGCAGCGGGAACTTCTTCCTTACTCGGTACTTGTCCACAGGTCCCAGGGGTCTTCCACGCAGCTTCTCAGCCTCCTGGTAGTGTGCTTCAAGCCACAGCGCCTGCAGCTTGGCGTGCGACTCCTTGGTGAACTTGTGGTTTTCCAGGATATGATAGAGCTCGCGGTAGTTGCCACCGTGAAAGGCCACGATGGCTCGTGCGCGTAGCACCGACTCATTCTTGTTGAGGGCCTCGCAGGCCGCAGGGGCCACGGGCAGCGACCAGAGGAAGCGACCCAGGCGCTCCACATCGCCGCTCTCTTCCAGGGTCTCACATACCCCGGCCACTTGCTGGGGGCTGAAATTCAAGATGGGCAGCTGGAACATCGAGGCAGTGCCGGCGGCGGCGAGGGCACTGAGCCCGGAGCGGGACACGCAGCAGATGCCCGCGGGCCTGGCTGAGTGTGCCAGGCGCTTGTTGATGAGGCGGATTGGGGCAGCGGGACACACTACCGGGTGGCGGCGGTGGCTCAACGGCCGGGAGCGGACGACCCCGACTGGAGGAGGTGCTGGCTCCGCGGCTTGGGGTTCGGGCTCGGCTCGGCTCAGGCACGCCGGCAGGCGAGCTGGCTCCGCTATTGGGGTGGGCGGATTGGCGGCGCGGGCGCCATGGAGACCCCCTGTCAGTCACCGGGTGGCTCTGCCAATCAAAGCTTGAATCAGCACGCCAGGACTATTTCAGCACCTCCCCGTGCTCGACAGCGCCCGCCG

The bisulfite treated sequence from SEQ ID NO: 3 is shown in SEQ ID NO:4 (Y represents C or U) as follows:

YGUTGGTTUUYGTGGTYGYGGUUAAAGGGYGUUAGTGAAGUUAGGUYGGUYGYGGGGTGGGGYGGGAAAYGGUAAUUYGAGUUYGYGGGTUUUTGGTUAUUTGGUTTUTGTGGYGUTGGATTTGUAGGTUTUTUATUTTUTTUUUUTUTTTUATTTTGTTTTTUTYGTTTTTAUAUTGGAATUTGUTTUTGAGUATUUTGGATGGGUAAUTUAGATGTYGUAUTYGUTGTYGUTGGAYGTGATGGAGATGGUTGAAGTGGUYGUUTTGUTGGATAGAUTGGYGGUYGGGUTGGTGGYGAYGUUUAGUAUUTUTGGYGTGUYGTYGUUUTUYGUUYGTAGTGUUYGUUYGGAAUUUTGTGAUAGGAUUTGUTGUTGGAGTUTAYGGGGAAGAAAGGGAAUAYGGGTUAUAGUTYGTAAUAUAGUYGUUYGTYGUUYGUUUUUAUUAUUTUUTGYGTUUUTUUAGUUYGGYGGUTAGAGGTTUTTUYGAAGAUUUUAGUAGTTAAGGYGAGGATTAUTYGGUAAGGYGAUAGAGGTYGGGUAGGAGTUYGYGGUYGUUUUTUYGYGGGTUUUTAUTGTTGTGUUUUAUUATGATTYGYGUUUTTTUTUAGYGUYGUAGUTUUUAGGTUTTTTATATAUTGGGUTTGUAGTGTUUTUUUTGTTTGTTGTGUAUTTTTGGGAGATTTAUAGGAUTGTUAGGAAATAAAYGAAGTGTGGGTGAUAAGGAUAGATYGGTUUUAGGGAUATGGGATAUUATTTUTAAYGGTGYGTUTGUUTGGGGUTTAYGUUTGAGUAAGTGUTUUYGYGATYGYGTTGGATYGGGTYGGUUYGAUUTAAAGGUAAAAAGGATTGTAAGGAGUATTTGGGUUTGGGGGATGAGGUYGGGUTUTTUYGUAAAATGTTTGGUTAATYGAGATUUAUAUTUYGGGAAAYGAAAAGUUTYGGUUUAYGGAUTAGTTTGUTGTTTTGAGAUAAGAGTGGUAGGUAGYGUATGGGAAGAGGAAAAUTYGAUTTUUUATTTUTUAAAAGTUUATTGAAAAUTGTTTTUAAAGUATTAGGGAGGGATATAUUTGGGUTTTGAAAATGATAATTAAUATGAAAUYGAUAUATTGUTAATAAGUUAGGUTTYGATTAATAGTAGGAGUUTGTGUUUAGGUTGAUAAGAATTAGUTTTGGGTUTTGGGGTUUUTAAUTGTTTGTAGGATTTTAATTTTTTATTTUTUTTUUUUTUTUYGTUUUAGTGAAGUUTTGGTTTGGTGAUYGGGAUUAGUTGUTGUAGUTGUUYGUAGGYGTGGGGAGUUAGYGGGTGGGGAAGUAGAGUYGGUAAGGGAAUTUUYGGUUAGGAGUUUAGAAYGUAGGGUTUTTAAUUUAAGAUAGAUYGYGUTUUUAAUTUUTGUTGAATTGGAGTYGUUAGGGAUTGGGTAAGGGYGUUAGAGGTGUUTUUAUUYGUUTUUTUUUYGGTGYGUTUAAAGYGYGGAGGUUTUTAGGTAUYGAUUTGTTUTTGGUTGUAGUYGUTYGGTUUUTTTGTYGGYGGTTTTTGAAUUAGTTGUUUAUUTGYGTAGGGGTUAGTUYGGTTGUUTGGGYGAGUTUAYGTTTTTTGUTGGGGTTAGGGTATGGATUUTGUAGGTAUUAUTYGYGTAGUAGGTGUYGYGTGYGUTUUTTGAAGUAGTGTGTUTTUTGTTYGUYGTUUUAAATGGTGYGYGGUAGYGGGAAUTTUTTUUTTAUTYGGTAUTTGTUUAUAGGTUUUAGGGGTUTTUUAYGUAGUTTUTUAGUUTUUTGGTAGTGTGUTTUAAGUUAUAGYGUUTGUAGUTTGGYGTGYGAUTUUTTGGTGAAUTTGTGGTTTTUUAGGATATGATAGAGUTYGYGGTAGTTGUUAUYGTGAAAGGUUAYGATGGUTYGTGYGYGTAGUAUYGAUTUATTUTTGTTGAGGGUUTYGUAGGUYGUAGGGGUUAYGGGUAGYGAUUAGAGGAAGYGAUUUAGGYGUTUUAUATYGUYGUTUTUTTUUAGGGTUTUAUATAUUUYGGUUAUTTGUTGGGGGUTGAAATTUAAGATGGGUAGUTGGAAUATYGAGGUAGTGUYGGYGGYGGYGAGGGUAUTGAGUUYGGAGYGGGAUAYGUAGUAGATGUUYGYGGGUUTGGUTGAGTGTGUUAGGYGUTTGTTGATGAGGYGGATTGGGGUAGYGGGAUAUAUTAUYGGGTGGYGGYGGTGGUTUAAYGGUYGGGAGYGGAYGAUUUYGAUTGGAGGAGGTGUTGGUTUYGYGGUTTGGGGTTYGGGUTYGGUTYGGUTUAGGUAYGUYGGUAGGYGAGUTGGUTUYGUTATTGGGGTGGGYGGATTGGYGGYGYGGGYGUUATGGAGAUUUUUTGTUAGTUAUYGGGTGGUTUTGUUAATUAAAGUTTGAATUAGUAYGUUAGGAUTATTTUAGUAUUTUUUYGTGUTYGAUAGYGUUYGUYG

Example 2. STAMP-EP1: Lung Cancer Cell Line Model Detection-BSP(Bisulfite Sequencing PCR) Detection

1. Genomic DNA was extracted from lung cancer cell line A549 and normallung fibroblast cell line MRC5;

2. The extracted genomic DNA of A549 and MRC5 cell lines were treatedwith bisulfite and used as templates for subsequent PCR amplification;

3. Primers were designed according to SEQ ID NO: 1. Four pairs ofprimers were designed for amplification of different sequence regions.The primer sequences and the detected methylation sites are shown inTable 1.

TABLE 1 Detected CpG site Primer Name 5′-3′ Primer Sequence No.STAMP-EP1-Sanger- AGGTTTTGTAAAATATTGAAGAAAAGTTTAGGAG CpG sites1-Primer-F (SEQ ID NO: 5) 001-059 STAMP-EP1-Sanger-CAACCTCCTAATAATATACTTCAAACCACAA 1-Primer-R (SEQ ID NO: 6)STAMP-EP1-Sanger- TATTTTGGAAAATTATAAGTTTATTAAGGAG CpG sites 2-Primer-F(SEQ ID NO: 7) 060-100 STAMP-EP1-Sanger-CAAAAATTAACTTTAAATCTTAAAATCCCTAAC 2-Primer-R (SEQ ID NO: 8)STAMP-EP1-Sanger- AATTTTTGTTAGTTTGGGTATAGGTTTTTATTAT CpG sites3-Primer-F (SEQ ID NO: 9) 101-147 STAMP-EP1-Sanger-ACCCTATAACAAAACCTACTACTAAAATCTAC 3-Primer-R (SEQ ID NO: 10)STAMP-EP1-Sanger- GAATTATGGTGGGGTATAATAGTAGGGATT CpG sites 4-Primer-F(SEQ ID NO: 11) 148-178 STAMP-EP1-Sanger-TCTAAACAAACRCTCAACTTACAATTCRAATTC 4-Primer-R (SEQ ID NO: 12)

4. After PCR amplification, 2% agarose gel electrophoresis was used todetect the specificity of the PCR fragments. The target fragments wererecovered by gel cutting and connected to T vector which was transformedinto competent Escherichia coli. The bacteria were spread on plate, andclones were selected the next day and sequenced. Ten clones wereselected for each fragment for Sanger sequencing.

5. The sequencing results are shown in FIG. 1. The average methylationvalue of lung cancer cell line A549 is 70.73%, while that in normal lungfiber cell line MRC5 is 0.45%.

6. The results showed that STAMP-EP1 was an excellent marker for lungcancer.

Example 3. STAMP-EP1: Lung Cancer—Clinical Sample Assay—Pyrosequencing

1. Clinical samples: 20 pairs of paracancerous-lung cancer samples wereobtained, with paracancerous samples as control group and lung cancersamples as experimental group;

2. DNA extraction: DNA was extracted from the experimental group and thecontrol group respectively. Phenol-chloroform extraction method was usedin this experiment, which is not limited thereto;

3. Bisulfite treatment: the extracted DNA samples were treated withbisulfite and the procedures were strictly followed. EZ DNAMethylation-Gold Kit (ZYMO Research, Cat #D5006) was used in thisexperiment, which is not limited thereto;

4. PCR amplification: the bisulfite treated samples were used as PCRtemplates, and PCR primers and pyrosequencing primers were designedbased on STAMP-EP1 sequences. Following PCR amplification, themethylation values of STAMP-EP1 were detected by pyrosequencing as themethylation values of sites 064, 065, 066, 067, 068 and 069. The PCRprimers sequences, pyrosequencing primers sequences, the detectingsequences of pyrosequencing and the detected sites are shown in Table 2;

TABLE 2 Primer Name 5′-3′ sequence Remark STAMP-EP1-Pyroseq-GAAGTATATTATTAGGAGGTTGAGAA The sites detected Primer-F G (SEQ ID NO: 13)by the primer pair: STAMP-EP1-Pyroseq- TACRCTCCTTAAAACAATATATCTTC064, 065, 066, 067, Primer-R(5′- (SEQ ID NO: 14) 068, 069 Biotin 

) STAMP-EP1-Pyroseq- CCCTGGGACCTGTGGACAAGT  Primer-Seq (SEQ ID NO: 15)Pyrosequencing ATYGAGTAAGGAAGAAGTTTTYGTTG detectingTYGYGTATTATTTGGGAYGGYG sequences (SEQ ID NO: 16)

5. Agarose gel electrophoresis: the specificity of PCR amplification wasidentified by agarose gel electrophoresis, and the size of the amplifiedfragment should be 139 bp;

6. Pyrosequencing: Pyro Mark Q96 ID pyrosequencing instrument (QIAGEN)was used for sequencing, and the procedures in instructions werestrictly followed;

7. Calculation of STAMP-EP1 methylation value: pyrosequencing can detectthe methylation values of sites 064, 065, 066, 067, 068, and 069 in thetarget region, and the average value were calculated as the STAMP-EP1methylation value in the sample;

8. Results analysis: the methylation value of STAMP-EP1 was comparedbetween the control group and the experimental group, as shown in FIG.2. It showed that among the clinical samples of lung cancer, theSTAMP-EP1 methylation value of the lung cancer experimental group wassignificantly increased, with P<0.0001, a sensitivity of 100% and aspecificity of 100%.

Example 4. STAMP-EP1: Colorectal Cancer—Clinical SampleAssay—Pyrosequencing

1. Clinical samples: 10 paracancerous clinical samples of colorectalcancer were used as the control group, and 28 colorectal cancer clinicalsamples were used as the experimental group;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3;

3. Results analysis: the methylation value of STAMP-EP1 was comparedbetween the control group and the experimental group, as shown in FIG.3. It showed that among the clinical samples of colorectal cancer, theSTAMP-EP1 methylation value of the colorectal cancer experimental groupwas significantly increased, with P<0.0001, a sensitivity of 100% and aspecificity of 100%.

Example 5. STAMP-EP1: Gastric Cancer—Clinical SampleAssay—Pyrosequencing

1. Clinical samples: 10 normal gastric (or gastritis) clinical sampleswere used as the control group, 10 gastric cancer resection edgeclinical samples were used as the experimental group 1, and 20 gastriccancer clinical samples were used as experimental group 2;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3; 8.Results analysis: the methylation value of STAMP-EP1 was compared amongthe normal gastric (or gastritis) control group, the resection edgeexperimental group 1 and gastric cancer experimental group 2, as shownin FIG. 4. The results showed that among the clinical samples of gastriccancer, the STAMP-EP1 methylation value of the gastric cancerexperimental group 2 was significantly increased, P<0.0001. Meanwhile,the methylation of resection edge experimental group 1 was between thenormal gastric (or gastritis) control group and the gastric cancerexperimental group 2, which indicated that the methylation of STAMP-EP1began to change at the resection edge. Therefore, as a marker of gastriccancer, STAMP-EP1 can be used to detect gastric cancer, and can also beused to determine the resection edge of gastric cancer.

Example 6. STAMP-EP1: Cervical Cancer—Clinical SampleAssay—Pyrosequencing

1. Clinical samples: 13 paracancerous clinical samples of cervicalcancer were used as the control group, and 26 cervical cancer clinicalsamples were used as the experimental group;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3;

8. Results analysis: the methylation value of STAMP-EP1 was comparedbetween the control group and the experimental group, as shown in FIG.5. It shows that among the clinical samples of cervical cancer, theSTAMP-EP1 methylation value of the cervical cancer experimental groupwas significantly increased, with P<0.0001, a sensitivity of 92.3% and aspecificity of 100%.

Example 7. STAMP-EP1: Liver Cancer—Clinical Sample Assay—Pyrosequencing

1. Clinical samples: 21 pairs of paracancerous-liver cancer samples wereobtained, with paracancerous samples as liver cancer control group andliver cancer samples as liver cancer experimental group;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3;

8. Results analysis: the methylation value of STAMP-EP1 was comparedbetween the control group and the experimental group, as shown in FIG.6. It shows that among the clinical samples of liver cancer, theSTAMP-EP1 methylation value of the liver cancer experimental group wassignificantly increased, with P<0.0001, a sensitivity of 100% and aspecificity of 100%.

Example 8. STAMP-EP1: Breast Cancer—Clinical Sample Assay—Pyrosequencing

1. Clinical samples: 22 pairs of paracancerous-breast cancer sampleswere obtained, with paracancerous samples as control group and breastcancer samples as experimental group;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3;

8. Results analysis: the methylation value of STAMP-EP1 was comparedbetween the control group and the experimental group, as shown in FIG.7. It shows that among the clinical samples of breast cancer, theSTAMP-EP1 methylation value of the breast cancer experimental group wassignificantly increased, with P<0.0001, a sensitivity of 100% and aspecificity of 100%.

Example 9. STAMP-EP1: Pancreatic Cancer—Clinical SampleAssay—Pyrosequencing

1. Clinical samples: 18 pairs of paracancerous-pancreatic cancer sampleswere obtained, with paracancerous samples as control group andpancreatic cancer samples as experimental group;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3;

8. Results analysis: the methylation value of STAMP-EP1 was comparedbetween the control group and the experimental group, as shown in FIG.8. It shows that among the clinical samples of pancreatic cancer, theSTAMP-EP1 methylation value of the pancreatic cancer experimental groupwas significantly increased, with P<0.0001, a sensitivity of 94.44% anda specificity of 100%.

Example 10. STAMP-EP1: Head and Neck Cancer—Clinical SampleAssay—Pyrosequencing

1. Clinical samples: 11 pairs of paracancerous-head and neck cancersamples were obtained, including 5 cases of laryngeal cancer, 2 cases oftonsil cancer, 2 cases of epiglottis cancer, 1 case of tongue basecancer, and 1 case of hypopharyngeal cancer. Paracancerous samples wereused as control group, and cancer samples as experimental group;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3;

8. Results analysis: the methylation value of STAMP-EP1 was comparedbetween the control group and the experimental group, as shown in FIG.9. It shows that among the clinical samples of head and neck cancer, theSTAMP-EP1 methylation value of the head and neck cancer experimentalgroup was significantly increased, with P<0.0001, a sensitivity of 100%and a specificity of 100%.

Example 11. STAMP-EP1: Gallbladder Cancer—Clinical SampleAssay—Pyrosequencing

1. Clinical samples: bile samples were obtained from 12 non-cancerpatients and 10 gallbladder cancer patients, with non-cancer samples ascontrol group and gallbladder cancer samples as experimental group;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3;

8. Results analysis: the methylation value of STAMP-EP1 was comparedbetween the control group and the experimental group, as shown in FIG.10. It shows that among the clinical samples of gallbladder cancer, theSTAMP-EP1 methylation value of the gallbladder cancer experimental groupwas significantly increased, with P<0.0001, a sensitivity of 90% and aspecificity of 100%.

Example 12. STAMP-EP1: Leukemia—Clinical Sample Assay—Pyrosequencing

1. Clinical samples: 10 non-leukemia bone marrow smear clinical sampleswere used as the control group, and 20 leukemia bone marrow smearclinical samples were used as the experimental group;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3;

8. Results analysis: the methylation value of STAMP-EP1 was comparedbetween the control group and the experimental group, as shown in FIG.11. It shows that among the clinical samples of leukemia, the STAMP-EP1methylation value of the leukemia experimental group was significantlyincreased, with P<0.0001, a sensitivity of 100% and a specificity of100%.

Example 13. STAMP-EP1: Renal Cancer—Clinical Sample Assay—Pyrosequencing

1. Clinical samples: 8 paracancerous clinical samples of renal cancerwere used as the control group, and 16 renal cancer clinical sampleswere used as the experimental group;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3;

8. Results analysis: the methylation value of STAMP-EP1 was comparedbetween the control group and the experimental group, as shown in FIG.12. It shows that among the clinical samples of renal cancer, theSTAMP-EP1 methylation value of the renal cancer experimental group wassignificantly increased, with P<0.0001, a sensitivity of 93.75% and aspecificity of 100%.

Example 14. STAMP-EP1: Bladder Cancer—Clinical SampleAssay—Pyrosequencing

1. Clinical samples: 5 cases of bladder cancer paracancerous samples andnon-cancer urine samples were used as the control group, and 7 cases ofbladder cancer tissue samples and bladder cancer urine samples were usedas the experimental group;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3;

8. Results analysis: the methylation value of STAMP-EP1 was comparedbetween the control group and the experimental group, as shown in FIG.13. It shows that among the clinical samples of bladder cancer, theSTAMP-EP1 methylation value of the bladder cancer experimental group wassignificantly increased.

Example 15. STAMP-EP1: Plasma Sample—Clinical Sample Assay

1. In order to prove that SATMP-C tumor marker can also be detectedusing liquid biopsy, 20 normal plasma samples were used as the controlgroup, and plasma samples from patients with different tumor types wereobtained, including 10 cases of liver cancer, 10 cases of pancreaticcancer, 10 cases of lung cancer, 10 cases of colorectal cancer, and 10cases of breast cancer;

2. Step 2, 3, 4, 5, 6, and 7 are the same as those in Example 3;

8. Results analysis: as shown in FIG. 14, compared with the normalplasma control group, the methylation values of liver cancer, pancreaticcancer, lung cancer, colorectal cancer and breast cancer groups weresignificantly increased.

Each reference provided herein is incorporated by reference to the sameextent as if each reference was individually incorporated by reference.In addition, it should be understood that based on the above teachingcontent of the disclosure, those skilled in the art can practice variouschanges or modifications to the disclosure, and these equivalent formsalso fall within the scope of the appended claims.

1-16. (canceled)
 17. A method for detecting tumor, wherein said methodcomprising: detecting the modification on the CPG site(s) of apolynucleotide by a tumor detection agent or kit, if thehypermethylation of a subject is detected, the subject can be identifiedas having a high-risk of tumor; said polynucleotide comprises: (a) thepolynucleotide with a nucleotide sequence as shown in SEQ ID NO: 1; (b)a fragment of SEQ ID NO: 1, containing the residues 1-589, the residues632-1218, the residues 1322-2066 or the residues 2100-2448 of SEQ ID NO:1; or (c) a nucleic acid complementary to the polynucleotide or fragmentof (a) or (b); wherein the tumors comprise: hematologic cancers such aslymphoma, multiple myeloma; digestive system tumors such as esophagealcancer, gastric cancer, colorectal cancer, liver cancer, pancreaticcancer, bile duct and gallbladder cancer; respiratory system tumors suchas pleuroma; nervous system tumors such as glioma, neuroblastoma,meningioma; head and neck tumors such as oral cancer, tongue cancer,laryngeal cancer, nasopharyngeal cancer; gynecological and reproductivesystem tumors such as breast cancer, ovarian cancer, cervical cancer,vulvar cancer, testicular cancer, prostate cancer, penile cancer;urinary system tumors such as kidney cancer, skin and other systemstumors such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroidcancer.
 18. The method according to claim 17, wherein samples of thetumor comprise: tissue samples, paraffin embedded samples, bloodsamples, pleural effusion samples, and alveolar lavage fluid samples,ascites and lavage fluid samples, bile samples, stool samples, urinesamples, saliva samples, sputum samples, cerebrospinal fluid samples,cell smear samples, cervical scraping or brushing samples, tissue andcell biopsy samples.
 19. The method according to claim 17, wherein saidtumor detection agent or kit is specifically detect the polynucleotide,or the Panel or gene group containing the polynucleotide.
 20. The methodaccording to claim 17, wherein said tumor detection agent or kitcomprises primers of probes.
 21. The method according to claim 20,wherein said primers are: the primers shown in SEQ ID NO: 5 and 6; theprimers shown in SEQ ID NO: 7 and 8; the primers shown in SEQ ID NO: 9and 10; the primers shown in SEQ ID NO: 11 and 12; or the primers shownin SEQ ID NO: 13 and
 14. 22. The method according to claim 17, whereinsaid modification comprises 5-methylation, 5-hydroxymethylation,5-formylcytosine (5fC) or 5-carboxylcytosine (5-caC).
 23. A method ofpreparing a tumor detection agent, comprising: providing apolynucleotide and designing a detection agent for specificallydetecting a target sequence which is the full length or fragment of thepolynucleotide; wherein said polynucleotide comprises: (a) thepolynucleotide with a nucleotide sequence as shown in SEQ ID NO: 1; (b)a fragment of SEQ ID NO: 1, containing the residues 1-589, the residues632-1218, the residues 1322-2066 or the residues 2100-2448 of SEQ ID NO:1; or (c) a nucleic acid complementary to the polynucleotide or fragmentof (a) or (b); wherein the tumors comprise: hematologic cancers such aslymphoma, multiple myeloma; digestive system tumors such as esophagealcancer, gastric cancer, colorectal cancer, liver cancer, pancreaticcancer, bile duct and gallbladder cancer; respiratory system tumors suchas pleuroma; nervous system tumors such as glioma, neuroblastoma,meningioma; head and neck tumors such as oral cancer, tongue cancer,laryngeal cancer, nasopharyngeal cancer; gynecological and reproductivesystem tumors such as breast cancer, ovarian cancer, cervical cancer,vulvar cancer, testicular cancer, prostate cancer, penile cancer;urinary system tumors such as kidney cancer, skin and other systemstumors such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroidcancer.
 24. The method according to claim 23, wherein the detectionagent specifically detect a gene sequence containing the targetsequence, and the gene sequence comprises a gene Panel or a gene group.25. The method according to claim 23, wherein the detection agentcomprises: primers or probes.
 26. The method according to claim 25,wherein the primers are: the primers shown in SEQ ID NO: 5 and 6; theprimers shown in SEQ ID NO: 7 and 8; the primers shown in SEQ ID NO: 9and 10; the primers shown in SEQ ID NO: 11 and 12; or the primers shownin SEQ ID NO: 13 and
 14. 27. A detection kit, comprising: container(s)and a detection agent in the container(s), said detection agentcomprises primers, the primers are: the primers shown in SEQ ID NO: 5and 6; the primers shown in SEQ ID NO: 7 and 8; the primers shown in SEQID NO: 9 and 10; the primers shown in SEQ ID NO: 11 and 12; or theprimers shown in SEQ ID NO: 13 and
 14. 28. A method of detecting themethylation profile of a sample in vitro, comprising: (i) providing thesample and extracting nucleic acid; (ii) detecting the modification onCPG site(s) of a target sequence in the nucleic acid of (i), wherein thetarget sequence is the polynucleotide comprises: (a) the polynucleotidewith a nucleotide sequence as shown in SEQ ID NO: 1; (b) a fragment ofSEQ ID NO: 1, containing the residues 1-589, the residues 632-1218, theresidues 1322-2066 or the residues 2100-2448 of SEQ ID NO: 1; or (c) anucleic acid complementary to the polynucleotide or fragment of (a) or(b).
 29. The method according to claim 28, wherein, in step (ii), theanalysis methods comprise pyrosequencing, bisulfite conversionsequencing, method using methylation chip, qPCR, digital PCR, secondgeneration sequencing, third generation sequencing, whole genomemethylation sequencing, DNA enrichment detection, simplified bisulfitesequencing technology, HPLC, MassArray, methylation specific PCR, ortheir combination, as well as in vitro detection and in vivo tracerdetection for the combined gene group of partial or all of themethylation sites in the sequence shown in SEQ ID NO:
 1. 30. The methodaccording to claim 28, wherein, step (ii) comprises: (1) treating theproduct of (i) to convert the unmodified cytosine into uracil; (2)analyzing the modification of the target sequence in the nucleic acidtreated by (1).
 31. The method according to claim 30, wherein treatingthe nucleic acid of step (i) with bisulfite.
 32. An isolatedpolynucleotide, wherein, the polynucleotide is converted from anoriginal polynucleotide, said original polynucleotide comprises: (a) afragment of SEQ ID NO: 1, containing the residues 1-589, the residues632-1218, the residues 1322-2066 or the residues 2100-2448 of SEQ ID NO:1; or (b) a nucleic acid complementary to the polynucleotide or fragmentof (a); and as compared with the sequence of the originalpolynucleotide, cytosine C of the CpG site(s) with modification isunchanged, and the unmodified cytosine is converted into T or U in theconverted polynucleotide.
 33. The polynucleotide according to claim 32,wherein treating the nucleic acid of the original polynucleotide withbisulfite to obtain the converted polynucleotide.